Plz, have an idea here too.

http://www.aaccnet.org/publications/cc/backissues/1983/documents/chem60_46.pdf

α -amylase facilitates the breakdown of starch to maltose; hydrolyzes bonds between glucose repeats. Carbohydrates are the key energy storage molecules used by living organisms. Plants store the energy of the sun in sugars using photosynthesis. Please visit http://www.worthington-biochem.com/aa/assay.html and or http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-a-amylase.html for procedure.

Hi, I think it depends on the amount of alpha amylase produced in seed sample during the germination, in some seeds like wheat, barely etc this amount is high, if the amount of a-amylase is high and measurable, you can use both spectrophotometric methods ie. Gram's iodine and DNS, but DNS method is more common, considering using spectrometer the calculated amylase activity in these methods won't be high and accurate, these methods are proper for differential comparisons between samples, please keep it in your mind, given that DNS shows the amount of released reducing sugars, and the crude extract itself (as an enzyme sample) may contain some reducing sugars, therefore read the samples and then subtract the values from the value of a substrate blank sample. Otherwise, in some cases It may cause significant errors in final results. Alternatively, you can set an HPLC method because of its higher sensitivity and accuracy.

Regard

Please check the PDF attachments.

Dear

I can add you some documents:

-ALPHA-AMYLASE ASSAY PROCEDURE (AMYLASE SD METHOD). Megazyme International Ireland 2015.

- McKie, V. A. & McCleary, B. V. (2015). A rapid, automated  method for measuring α-amylase in pre-harvest sprouted (sprout damaged) wheat. Journal of Cereal Science, 64, 70-75.

I hope it will be helpful

Marius

https://secure.megazyme.com/files/Booklet/K-AMYLSD_DATA.pdf

https://secure.megazyme.com/Alpha-Amylase-Assay-Kit

http://www.aaccnet.org/publications/cc/backissues/1983/documents/chem60_46.pdf

http://www.aaccnet.org/publications/cc/backissues/1960/Documents/chem37_218.pdf

https://www.ncbi.nlm.nih.gov/pubmed/12374409

Kindly see here too..

http://www.aaccnet.org/publications/cc/backissues/1983/documents/chem60_46.pdf

Dear experts, Thank you very much for your response.

Dear Mabrouka,

I want to know which method did you use at last for alpha amylase assay in lettuce in your experiment? Have you done it on seeds or seedlings?

For measuring the amylase activity with DNS solution (Miller Method):

1. Prepare starch solution (10 mg/ml)

2. Prepare enzyme extract filtered with syringe filter

3. Prepare phosphate buffer solution (o.1(M), pH=7)

4. Add 1(ml) enzyme extract to test tube, then add 1(ml) of starch solution (it can be considered as a main sample)

5. Add 1(ml) enzyme extract to test tube, then add 1(ml) phosphate buffer (it can be considered as a blank or control sample)

6. Incubation of main and control sample at 37˚C for 30(min)

7. Add 2(ml) DNS solution to each test tube.

8. Transfer the tube to the water bath at boiling temperature for 15(min)

9. Keep the test tube at room temperature to cool the sample

10. Transfer the solution to the cuvette

11. Measure the absorption of the main sample at λ=540(nm)

It is necessary to mention that for calculating the enzyme activity the standard curve must be prepared by maltose solution.

Mina Karimi-Avargani Should I follow the same procedure for standard curve preparation?

Dear Monalisa,

For preparing standard curve, different concentration of maltose solution must be prepared. Then follow exactly according to the same procedure for enzyme assay (Substrate+ DNS). It is not necessary to add enzyme solution.